Application Note
Purification of metagenomic DNA from environmental water samples with theMasterPure™ Complete Kit
Introduction

Filtration, Lysis, and Protein Precipitation

Environmental water samples often harbor a complex array of

Each purification will use 1.5 mL of PBS with 0.1% Tween 20. Before

microorganisms. Metagenomic analysis of such samples is complicatedstarting, prepare enough buffer for the number of samples you will beby the relatively large sample processing volume and the unculturable

purifying by adding 1.5 μL of Tween 20 per 1.5 mL of PBS to achieve a finalnature of most environmental bacteria. The MasterPure™ Complete DNA

concentration of 0.1% Tween 20.

and RNA Purification Kit can be easily adapted to purify metagenomic
DNA from water samples. The isolated, randomly sheared DNA is highmolecular weight (~40 kB) and suitable for downstream applications
including next gen sequencing, PCR, and fosmid library production.
For increased reliability and simplicity in fosmid library production, weNote: It is critical to follow the recommended centrifugal speeds, asindicated in the protocol, for efficient recovery of the microbes from thewater samples.
1.

To remove debris from the collected water sample (100 mL), pourrecommend using the isolated DNA in conjunction with the CopyControl™

the water through Miracloth filtration material or sterile cheesecloth.

Fosmid Library Production Kit or CopyControl HTP Fosmid Library

Alternatively, centrifuge the sample at 1,000 × g for 5 minutes. Collect

Production Kit. For next gen sequencing and PCR applications, we

the water in a sterile container.

recommend using the NxSeq® UltraLow DNA Library Kit and FailSafe™
PCR Systems, respectively.

2.

an appropriate filter apparatus in order to trap the microbial mass on
the filter. Retain the filter membrane.

Methods
Use the reagents from the MasterPure Complete DNA & RNA Purification

Filter the water (100 mL) through the 0.45 μm filter membrane using

3.

Using forceps and scissors presoaked in 70% ethanol, remove themembrane from the filter apparatus, cut the membrane in half, andKit. You will need to supply the following additional reagents:

place each half (rounded side down) along the side (near the bottom)• Phosphate-buffered saline (PBS)of a 50 mL sterile conical tube. The upper surface of the filter needs• Ready-Lyse™ Lysozyme Solution (Lucigen Cat. No. R1804M)

to face the center (not the wall) of the tube. Do not allow the filter• 0.45-μm filter membranes (e.g., Sterlitech Cat. No. CA04547100)

membrane to dry out.

• Filtration apparatus (Millipore Sigma Cat. No. XX1004700, or4.

equivalent)

1.5 mL of PBS immediately before use. Add 1.5 mL of Filter Wash
Buffer containing 0.1% Tween 20 to the filter pieces in the tube.

• Miracloth filtration material (Millipore Sigma) or sterile cheesecloth• 50 mL conical tubes

5.

• 1.7 mL microcentrifuge tubes• Tween® 20 (molecular biology grade)

Prepare the Filter Wash Buffer by adding 1.5 μL of Tween 20 toVortex the tube at the low speed setting to rewet the filter pieces,then increase the setting to the highest speed.

6.

Transfer the cell suspension to a clean microcentrifuge tube, then• Isopropanol

centrifuge the tube at 14,000 × g or top speed in a microcentrifuge• 70% ethanol (prepared from absolute ethanol)for 2 minutes to pellet the cells. Discard the supernatant.

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lucigen.com | ph 888 575 9695 | f 608 831 9012 | United StatesISO 13485 Certified. For research or investigational use only.

7.

Resuspend the cell pellet in 300 μL of TE Buffer, then add 2 μL of

19. Add 500 μL of 70% ethanol without disturbing the pellet. Centrifuge

Ready-Lyse Lysozyme Solution and 1 μL of RNase A to the cellfor 5 minutes at 20,000 × g, or maximum speed, in a microcentrifuge

suspension. Mix, and centrifuge briefly.

at 4°C.

8.

Incubate the tube at 37°C for 30 minutes.

9.

Add 300 μL of 2X Tissue and Cell Lysis Solution and 1 μL ofProteinase K to the tube. Mix by vortexing.

10. Briefly pulse-centrifuge the tube to ensure that all of the solution is in
the bottom of the tube.

20. Use a pipet tip to remove the ethanol without dislodging the DNApellet. Briefly pulse-centrifuge the sample and remove any residualliquid with a pipet tip without disturbing the pellet.
21. Air-dry the pellet for 8 minutes at room temperature.
Note: Do not over-dry the DNA pellet.

11. Incubate at 65°C for 15 minutes.

22. Resuspend the DNA pellet in 40 μL of TE Buffer.

12. Place on ice for 3–5 minutes.

23. Validate the size and concentration of the isolated DNA by

13. Add 350 μL of MPC Protein Precipitation Reagent to the tube and mix
by vortexing vigorously for 10 seconds.
14. Pellet the debris by centrifuging for 10 minutes at 20,000 × g, ormaximum speed, in a microcentrifuge at 4°C.
15. Transfer the supernatant to a clean 1.7 mL microcentrifuge tube andelectrophoresis on a 1% agarose gel.
The isolated DNA is ready for PCR, next gen sequencing, or end-repair
and subsequent cloning into a vector for construction of a fosmid library.
If using the CopyControl Fosmid Library Production Kit, proceed with
end-repair (Section B) and ligation (Section E) in the supplied protocol(#MA171E: CopyControl Fosmid Library Production Kits).

discard the pellet.
16. Add 570 μL of isopropanol to the supernatant. Mix by inverting thetube several times.
17. Pellet the DNA by centrifuging for 10 minutes at 20,000 × g, ormaximum speed, in a microcentrifuge at 4°C.
18. Use a pipet tip to remove the isopropanol without dislodging theDNA pellet. Briefly pulse-centrifuge the sample, and remove any
residual liquid with a pipet tip without disturbing the pellet.

Lucigen Corporation

lucigen.com | ph 888 575 9695 | 608 831 9011 | United States
ISO 13485 Certified. For research or investigational use only.

NxSeq is a registered trademark, and CopyControl, FailSafe, MasterPure, andReadyLyse are trademarks of Lucigen.
Tween is a registered trademark of Croda International PLC.